Cell Tissue Res (1982) 226:441-447 Cell and Tissue Research �9 Springer-Verlag 1982

Further freeze-fracture studies on the uterine epithelium of Salamandra salamandra (L.) (Amphibia, Urodela) using the antibiotic filipin

Hartmut Greven and Horst Robenek*

Zoologisches Institut und Medizinische Cytobiologie der Universit/it Mtinster, MOnster, Bundes- republik Deutschland

Summary. The apical portion of the uterine lining of the ovoviviparous fire salamander, Salamandra salamandra, was studied by the freeze-fracture technique in conjunction with the polyene antibiotic filipin. Filipin-sterol complexes were found in the luminal plasmalemma and in the membranes limiting the mucous secretory granules typical of this epithelium. In all females, but particularly in non-pregnant females, more or less discrete clusters of filipin- sterol complexes were occasionally found overlying heavily affected secretory granules. The findings are discussed with regard to comparable results (Orci et al. 1980) based on the examination of collapsed and stretched urinary bladders of toads.

Key words: Uterine epi thel ium- Freeze-fracture- Filipin - Sterol heterogeneity

Zusammenfassung. Das apikale Plasmalemm und die Membranen der Sekret- grana im Uterusepithel des Feuersalamanders, Salamandra salamandra, sind nach Behandlung mit Filipin im Gefrierbruch unterschiedlich stark markiert. Bei tr/~chtigen und nichttr/~chtigen Weibchen - hier wahrscheinlich vermehrt - finden sich gelegentlich Felder von Filipin-Sterol Komplexen im apikalen Plasmalemm unmittelbar unter ebenfalls meist stark markierten Sekretgrana. Die Befunde werden im Hinblick auf im wesentlichen vergleichbare Beobachtungen an kollabierten und gedehnten Harnblasen yon Kr6ten (Orci et al. 1980) diskutiert.

The mono-layered uterine lining of the ovoviviparous urodelan species Salamandra salamandra represents a rather simple transporting epithelium. It consists of only one cell type, which is characterized by numerous mucous secretory granules,

Send offprint requests to: PD Dr. Hartmut Greven, Zoologisches Institut der Universit/it Miinster, H0fferstr. 1, D-4400 MOnster (Westf.), Federal Republic of Germany

* We are indebted to Mrs. K. Ott for excellent technical assistance, to Miss E.S. MacLure for linguistic corrections and to Dr. J.E. Grady of the Upjohn Co., Kalamazoo, Michigan USA, for kindly providing the filipin

0302-766X/82/0226/0441/$01.40

442 H. Greven and H. Robenek

mostly arranged beneath the luminal plasmalemma and varying in size and number from cell to cell. The general ultrastructure and perhaps the secretory activity of the epithelium does not seem to be influenced significantly by the reproductive cycle (Lostanlen et al. 1976; Greven 1977, 1980a; Greven and Robenek 1980a); only apicolateral tight junctions between adjacent cells show some minor alterations caused by the mechanical stress of the tissue during pregnancy (Greven and Robenek 1980b).

Using the freeze-fracture technique we found nearly the same density of intramembranous particles in the luminal plasmalemma of both pregnant and non- pregnant uteri orS. salamandra (Greven and Robenek 1980a). In the present study we have again examined the apical portion of this epithelium, in particular the luminal plasmalemma and the membranes limiting the secretory granules, by freeze-fracture in conjunction with the antibiotic filipin. This substance reacts specifically with cholesterol or other related 3-/3-hydroxysterols to produce specific membrane lesions; it can thus be used to demonstrate cholesterol distribution in biomembranes (e.g. Norman et al. 1976; Elias et al. 1979; Robinson and Karnovsky 1980; Friend and Bearer 1981; Bittman et al. 1981).

Materials and methods

Pregnant and non-pregnant females of Salamandra salamandra were sacrificed. Small pieces of uterine tissue were fixed for several hours in 2.5 ~ glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.3. After fixation the tissue was treated with the same buffer containing 0.2~o filipin and 1% dimethylsulfoxide for 1 h at room temperature in the dark.

Tissue was then rinsed briefly in buffer and soaked in 30 % glycerol. After glycerination it was frozen in liquid Freon 22 at - 150 ~ C and stored in liquid nitrogen. Replicas were made according to Moor and Mtihlethaler (1963) in a Balzers BA 300 freeze-etching apparatus (Balzers AG, Liechtenstein) and examined using a Philipps 201 electron microscope operating at 60 kV.

All freeze-fracture micrographs are printed so that the direction of shadowing is towards the top of the page.

Results

The areas obtained from apical plasmalemmata of both pregnant and non- pregnant uteri by freeze-fracture were relatively small and unevenly studded by filipin-sterol complexes. In most cases membranes affected by filipin displayed randomly distributed protuberances and depressions about 30 nm in diameter, apparently in both the protoplasmic face (P-face) and the external face (E-face). The density of the filipin-sterol complexes, which ranged from very few to numerous in the membrane areas fractured (Fig. I a, b) varied not only from cell to cell but also within the luminal plasmalemma of a single cell. In some cases we observed more or less discrete clusters of lesions, often situated on bulges of the luminal plasmalemma (Fig. 1 b). Membrane areas between these clusters exhibited only a few lesions. Clustering appeared to be somewhat more frequent in non- pregnant females, but the limited material available did not allow statistically significant counts to be performed.

A random distribution of filipin-sterol complexes was also found in the limiting membrane of the secretory granules (Fig. 2a-e). Often the membranes of granules aligned immediately beneath the plasmalemma or even in more basal parts of the

Freeze-fracture on the uterus of Salamandra using filipin 443

Fig. 1 a, b. Apical plasmalemma of the uterine epithelium in S. salamandra, a Pregnant female. Note the heavily affected plasmalemma (big arrow) and the membranes limiting the secretory granules (arrowhead). x 20,000. b Non-pregnant female. Clusters (arrowheads) of filipin-sterol complexes are visible, lu lumen of the uterus, x 30,000

cells were heavily affected, whereas others in the immedia te proximi ty were not. Unfor tuna te ly we have no clear images showing the fracture plane s tepped f rom the apical m e m b r a n e just overlying a granule to the limiting m e m b r a n e o f the granule itself. We conclude, however, f rom cross and oblique fractures where m e m b r a n e s overlying a granule are often heavily affected (Fig. 2a, b, e) that clusters o f filipin-

444 H. Greven and H. Robenek

Fig. 2a-e. Secretory granules (a, b) and presumed sites of exocytosis (c--e) in the uterine epithelium of S. salamandra, a Heavily affected secretory granules and similarly labelled plasmalemma overlying the granules (arrowheads) in a pregnant female. Discharged granule (asterisk). x 28,000. b Similar features seen at higher magnification, x 45,000. c Ongoing exocytosis (arrowhead), pregnant female. • 60,000. d As before. Non-pregnant female. Most of the granules are unlabelled, x 45,000. e Site of exocytosis obscured by filipin-sterol complexes (arrowhead). lu lumen of the uterus, x 60,00

Freeze-fracture on the uterus of Salamandra using filipin 445

sterol complexes are localized in those regions of the plasmalemma which overlie granules.

Images showing structures which could be interpreted as exocytosis stages were rarely seen in our material and were often obscured by filipin-sterol complexes (Fig. 2c, d, e). Small hollows or crater-like depressions which could indicate the initiation of exocytotic events are occasionally observed in areas free of filipin- induced lesions as well as in filipin-untreated material (unpublished data). This phenomenon was perhaps found more frequently in pregnant females. Discharged granules appeared as pockets or infoldings of the luminal surface (Fig. 2 a). In a few instances areas of lateral membranes with tight junctions were exposed; membrane areas between junctional strands lacked filipin-sterol complexes.

Discussion

The results of the present study are to some extent difficult to interpret at the present time. Filipin is now established as being a reliable specific reagent for localizing and quantifying sterols in biomembranes and may even be used as a permeability tracer (e.g. Bittman et al. 1981; Friend and Bearer 1981).

Inhomogenous labelling of biomembranes with filipin indicates a nonuniform distribution of cholesterol and has been reported in numerous studies of a variety of tissues (e.g. Elias et al. 1979; Orci et al. 1980, 1981 and others). Such regional differences may also be expected according to biochemical investigations (for review see Demel and Kruijff 1976; Norman et al. 1976). Although in some tissues an insufficient penetration of the substance should be taken into consideration due to various junctions or perhaps to the cell coat (for some discussion of this topic see Elias et al. 1979; Warren and Severs 1980; Friend and Bearer 1981), in our experiments filipin could reach all sites of interest. In such cases heterogenous labelling of membranes has been suggested as representing real differences in cholesterol distribution (Orci et al. 1980, 1981; Elias et al. 1979; see also Robenek and Greven 1981 and others).

Clusters of filipin-induced lesions, however, do not seem to be fortuitous. They were a reproducible pattern reflecting a high content of cholesterol at these sites. Principally these findings correspond with those described by Orci et al. (1980), who also observed clusters of filipin-sterol complexes in the apical membrane overlying the secretory granules of granular cells in the unstretched urinary bladders of toads. These clusters were, however, more discrete than in our material. In stretched bladders distribution of filipin-induced lesions were more homogeneous. The authors speculated that 1) the secretory granule membrane is cholesterol-rich when compared to much of the apical plasma membrane, 2) the clusters of filipin-sterol complexes are associated with sites of prospective or ongoing exocytosis and 3) sites of interaction between granule and luminal membranes might perhaps be enriched in cholesterol as the result of incorporation of cholesterol-rich granule membrane into the apical membrane. The significance of all these observations is unclear at the present time (see also Orci et al. 1981). It should be noted, however, that cholesterol influences membrane properties such as fluidity, permeability and enzymatic activity (Jain 1975; Demel and Kruijff 1976; Norman et al. 1976; Bittman et al. 1981) and "fluid" membranes of large coated pits free of filipin-cholesterol complexes have been interpreted as modifications to facilitate endocytosis

446 H. Greven and H. Robenek

(Montesano et al. 1979). Our own observations on the uterine lining of S. salamandra only show that clustering of filipin-sterol complexes in membranes overlying secretory granules and heavy labelling of membranes of secretory granules may be a widespread phenomenon.

Nevertheless, there might be some other interesting parallels between the uterus of the salamander and the urinary bladder of the toad. Granular cells of the latter also release their secretory granules in response to artificial stretching without any hormonal stimulus (Brown et al. 1981), in which case clustering of filipin-sterol complexes disappears (Orci et al. 1980). In our material such differences are not so significant. This is probably due to the fact that stretched (pregnant) uteri collapse somewhat after the larvae are removed. The stimulus for releasing the secretory product into the lumen is unknown. It may be hormonal, mechanical or even both. Differences in secretory activity between pregnant and non-pregnant uteri were not entirely excluded in a previous paper (Greven 1977). Although counts were not made, the observation of apparently more interactions of luminal plasma membranes and secretory granules as seen in freeze-fracture replicas of filipin- untreated pregnant uteri (unpublished data) may indicate more frequent exocytoses in pregnant females caused perhaps by mechanical stress. It should be noted that the uterus of S. salamandra is only a slightly modified part of the oviduct, which is provided with different glands along its entire length which secrete the egg jelly (Greven 1980). There is evidence that, at least in Anura, jelly secretion during oviposition is hormonally regulated, but in both Anura and Urodela a mechanical stimulus exerted by the passing eggs is also involved (Thornton and Evennett 1969; Jorgensen and Vijayakumar 1970).

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Freeze-fracture on the uterus of Salamandra using filipin 447

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Accepted June 7, 1982